Voltage-dependent anion channel 1 (VDAC1; mitochondrial porin) is a pore-forming protein found in the outer mitochondrial membrane in all eukaryotic cells. VDAC1 controls cross-talk between mitochondria and the rest of the cell by serving as a gatekeeper for the entry and exit of metabolites. In addition to regulating metabolic and energetic functions of the cell, VDAC1 also provides a point of convergence for a variety of cell survival and death signals, mediated via its association with various ligands and proteins. Moreover, VDAC1 is a key player in mitochondria-mediated apoptosis, participating in the release of mitochondria pro-apoptotic proteins (e.g. cytochrome c, AIF and Smac/DIABLO) to the cytosol and interacting with apoptosis regulatory proteins such as Bcl-2, Bcl-X and hexokinase (HK).
Three mammalian isoforms of VDAC are known, VDAC1, VDAC2 and VDAC3, where VDAC1 is the major isoform expressed in mammalian cells. Blachly-Dysion et al. (Blachly-Dyson E et al., 1993. J Biol Chem. 268(3):1835-41) disclosed the cloning and functional expression in yeast of two human VDAC isoforms, VDAC1 and VDAC2. U.S. Pat. No. 5,780,235 discloses two VDAC sequences, which were named HACH (Human voltage-dependent Anion Channel), subsequently identified as VDAC2 and VDAC3. That patent provides genetically engineered expression vectors, host cells containing the vector, a method for producing HACH and a method for identifying pharmaceutical compositions inhibiting the expression and activity of HACH and for the use of such compositions for the treatment of cancer and proliferative diseases.
Computer modeling of the VDAC's primary amino acid sequences led to the development of models showing the transmembrane organization of VDAC1, consisting of a single amphipathic N-terminal α-helix and 13 or 16 transmembrane β-strands. These β-strands are connected by several peptide loops of different sizes on both sides of the membrane that serve as potential protein interacting sites. Recently, the three-dimensional structure of isoform 1 of VDAC was determined at atomic resolution by three independent technical approaches, leading to a structure featuring a 19-stranded β-barrel and an N-terminal α-helical region located inside the pore.
Several functional implications of this architecture were suggested. It was proposed that the N-terminal region lies inside the pore yet could move in the open space. The mobility of the N-terminal region was further supported by studies showing that the N-terminal α-helix exhibits motion during voltage-gating, and that anti-VDAC1 antibodies raised against the N-terminal region of the protein interact with membranal VDAC1. It has been also shown that the N-terminal α-helix interacts with apoptosis-regulating proteins of the Bcl-2 family (i.e., Bax, Bcl2 and Bcl-xL) and hexokinase (see, for example, Abu-Hamad S et al. 2009. J Cell Sci 122:1906-1916; Shi Y et al. 2003. Biochem Biophys Res Commun 305:989-996; Arbel N et al. 2012. J Biol Chem. 287(27):23152-61, Arbel N and Shoshan-Barmatz V. 2010. J Biol Chem 285:6053-6062; Geula S et al. 2012. Biochem J. 15:444(3):475-485) and that movement of this VDAC1 segment out of the pore modulated the anti-apoptotic activities of these binding partners. Moreover, cells expressing N-terminal domain-truncated VDAC1 are resistant to apoptosis (Abu-Hamad S et al. 2009, ibid). These findings indicate that the N-terminal domain is required for apoptosis induction, interactions with HK-I, HK-II, Bcl-xL and Bcl-2 and for protection against apoptosis.
The GXXXG motif has been associated with dimerization of proteins including glycophorin A, human carbonic anhydrase, yeast ATP synthase and more. In VDAC1, the GXXXG motif is present in the N-terminus of the channel that forms the α-helix structure.
It was reported that in VDAC1, a glycine-rich sequence (GXXXG), highly conserved in mammals, connects the N-terminal domain to the β-barrel, thus providing the flexibility needed for N-terminal translocation in and out of the pore. GXXXG motif of the VDAC1 N-terminal domain is involved in N-terminal domain translocation out of the VDAC pore and in the oligomerization of VDAC1 (Geula S. et al., 2012, ibid) U.S. Pat. No. 8,440,788 to the inventor of the present invention and co-workers discloses that VDAC1 derived peptides having a point mutation within the GXXXG motif are capable of inhibiting apoptosis.
Apoptosis, also known as programmed cell death, plays a central role in, inter alia, development, immune cell regulation and tissue homeostasis in multicellular organisms. Genetic and molecular analysis from various species has indicated that the apoptotic pathway is highly conserved. In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing cancer. Mitochondria play an important role in the regulation of apoptotic cell death. The release of apoptogenic intermediates such as cytochrome c from the intermembranal space into the cytoplasm of a cell initiates a cascade of caspase activation that executes the cell death program. Substantial evidence links VDAC1 to apoptosis and suggests that VDAC1 is a critical player in the release of apoptogenic proteins from mitochondria in mammalian cells (Shoshan-Barmatz V and Gincel D. 2003. Cell Biochem Biophys 39: 279-292; Shoshan-Barmatz V et al. 2010. Molecular Aspects of Medicine 31(3):227-286; Shoshan-Barmatz V and Ben-Hail D. 2012. Mitochondrion 12(1):24-34; Shoshan-Barmatz V and Golan M. 2012. Current Medicinal Chemistry 19(5):714-35).
Diverse intrinsic cell death signals emanating from various subcellular organelles can induce the release of cytochrome c from mitochondria. The Bcl-2 family of pro- and anti-apoptotic proteins constitutes a decisive control point for apoptosis. Proteins in the Bcl-2 family are major regulators of apoptosis (reviewed in Kim R. 2005. Biochem Biophys Res Commun 333(2):336-43). Members of this family include both pro- and anti-apoptotic proteins and share homology in up to four conserved regions termed Bcl-2 homology (BH) 1-4 domains. The family can be divided into three main sub-classes: anti-apoptotic proteins, pro-apoptotic proteins and BH3-only proteins. The anti-apoptotic proteins, including hexokinase-I (HK-I), Bcl-2 and Bcl-xL, share homology throughout all four BH domains. The pro-apoptotic proteins can be further subdivided and include multi-domain proteins, such as Bax and Bak, which possess sequence homology in BH1-3 domains.
The more distantly related BH3-only proteins appear to be only pro-apoptotic and share sequence homology within the BH3 region, which is required for their apoptotic function. The BH3-only proteins include, for example, BID, NOXA, PUMA and BAD.
It is currently held that anti-apoptotic members of the Bcl-2 family of proteins, such as HK-I, HK-II, Bcl-2 and Bcl-xL, act to promote cell survival by interacting with VDAC. Conversely, pro-apoptotic members of the Bcl-2 family of proteins, including Bak and Bax, may interact with VDAC to promote release of cytochrome c. Because of the pivotal role that mitochondria play in apoptotic cell death, mitochondrial proteins serve as potential targets for apoptosis regulating therapies.
One major obstacle in cancer chemotherapy is inherent, or acquired, resistance, apparently due to the suppression of apoptosis in the cancerous cells. Hexokinase-I (HK-I) is an anti-apoptotic mitochondrial protein that binds to VDAC. Many tumor cells exhibit a high glycolytic rate, which is correlated with a high level of HK-I expression. It is believed that the overexpression of anti-apoptotic proteins such as HK-I in cancer cells is a self-defense mechanism of those cells and is related to the cell's resistance to chemotherapy. In B-chronic lymphocytic leukemia (CLL), the failure of mature B cells to undergo apoptosis constitute the primary cellular defect leading to the cancer disease (Montserrat E and Moreno C. 2008. Ann Oncol 2008 Sep. 19, Suppl 7:vii320-325).
The anti-apoptotic proteins of the Bcl-2 family, such as Bcl-2, Bcl-xL and Mcl-1 and XIAP are overexpressed in CLL, whereas the pro-apoptotic protein Bax in under-expressed (Kitada S et al. 1998. Blood 91:3379-3389).
Certain compositions related to VDAC1 and use thereof for either inhibiting or inducing apoptosis are known in the art. U.S. Patent Application Publication No. 2005/0085420 discloses methods of inhibiting apoptosis by promoting formation of a BAK/VDAC2 complex, and methods of promoting apoptosis by disrupting formation of a BAK/VDAC2 complex. The VDAC2/BAK inhibitor compound is, for example, a BH3 domain peptide, a BH3 domain-only mutein, an anti-VDAC2 antibody, a VDAC2 mutein and the like.
U.S. Patent Application Publication No. 2005/0234116 discloses small molecule compounds with utility as VDAC regulators, in particular as apoptosis suppressors.
U.S. Pat. Nos. 8,119,601 and 8,648,045 to the inventor of the present invention and others disclose VDAC variants and VDAC derived peptides as well as polynucleotides encoding same useful in inducing or regulating apoptosis and to pharmaceutical compositions comprising same useful in the treatment of diseases associated with aberrant apoptosis.
A publication of the inventor of the present invention and co-workers, published after the priority date of the present invention, demonstrates that VDAC1-based peptides, including short peptides selectively induced cells death of peripheral mononuclear cells (PBMC) from CLL patients while exhibiting minor effects on PBMCs from healthy donor (Prezma T et al. 2013 Sep. 19; 4:e809. doi: 10.1038/cddis.2013.316).
There remains an unmet need for improved therapeutic agents that specifically induce cancer cell apoptosis, particularly for improved peptides with improved pharmacokinetic characteristics for inducing cancer cell death.